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Include non-laxy links to extra MultiQC reports
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‎04-04-Pipeline-fastqc.Rmd‎

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@@ -76,8 +76,11 @@ What happens to the adapter content and sequence length distribution ?
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#### {-}
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Here's our [example RNAseq run](https://laxy.io/#/job/1qUtszdMrQnXC4tawunu92/?access_token=7ad22813-fa68-4848-b93e-de30d53a35b3) without a trimming step. For this dataset,
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important QC metrics like the "% Aligned" to the reference genome are very close, but not identical between trimmed and untrimmed reads.
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Here's a the MultiQC report for the example dataset with
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<a href="files/multiqc/SRP062287/multiqc_report.html" target="_blank">trimming</a> and
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<a href="files/multiqc/SRP062287_untrimmed/multiqc_report.html" target="_blank">excluding the trimming step</a>
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(and <a href="https://laxy.io/#/job/1qUtszdMrQnXC4tawunu92/?access_token=7ad22813-fa68-4848-b93e-de30d53a35b3" target="_blank">the full untrimmed run on laxy.io</a>).
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For this dataset, important QC metrics like the "% Aligned" to the reference genome are very close (within ~1%), but not identical between trimmed and untrimmed reads.
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> Trimming _is_ beneficial if you are _de novo_ assembling RNA-seq reads (eg, for a non-model organism), or using RNA-seq for variant calling.
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FastQC reports "Per base sequence content".
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![FastQC 'Per base sequence content' plot showing nucleotide composition bias in the first ~12 bases](images/multiqc/fastqc_per-base-sequence-content.png){width="100%"}
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![](images/multiqc/fastqc_per-base-sequence-content.png){width="100%" fig-alt="FastQC 'Per base sequence content' plot showing nucleotide composition bias in the first ~12 bases"}
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It is quite common to see some sequence bias at the 5' end of reads in RNA-seq libraries.
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FastQC shows plots summarizing exact duplicate reads, and overrepresented sequences.
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<a href="https://api.laxy.io/api/v1/job/3pLfQoLEuWeAnWh4H3Vvbv/files/output/results/fastqc/SRR2155413_fastqc.html?access_token=e0124ee9-c8ad-4164-b59f-ca2ae0ce4d2a#M8" target="_blank">SRR2155413 Sequence Duplication Levels</a>
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<a href="files/fastqc/SRP062287/untrimmed/SRR2155413_fastqc.html#M8" target="_blank">SRR2155413 Sequence Duplication Levels</a> (or
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<a href="https://api.laxy.io/api/v1/job/3pLfQoLEuWeAnWh4H3Vvbv/files/output/results/fastqc/SRR2155413_fastqc.html?access_token=e0124ee9-c8ad-4164-b59f-ca2ae0ce4d2a#M8" target="_blank">on laxy.io</a>).
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> Look at the Sequence Duplication Level plot - what might the peak at >10 duplication level represent ?
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> The red line represents the proportion that those same sequences would contribute after deduplication.

‎04-05-Pipeline-mapping-and-qc.Rmd‎

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For FFPE preserved tissue samples the mapping rate can be highly variable depending on sample quality.
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If the overall percentage of reads mapped is very low (<25% ?), there is chance you've chosen the wrong organism for your reference genome.
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[Here's an example](https://laxy.io/#/job/20nDhlhpb8xv53x3R2CACg/?access_token=29ddd348-0a55-4596-b6f9-060e5186236f) of attempting to use
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Here's <a href="files/multiqc/SRP062287_mouse_ref/multiqc_report.html" target="_blank">an example</a>
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(or <a href="https://laxy.io/#/job/20nDhlhpb8xv53x3R2CACg/?access_token=29ddd348-0a55-4596-b6f9-060e5186236f" target="_blank">on laxy.io</a>) of attempting to use
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the mouse genome with our human cell line example dataset.
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### MultiQC: Duplication statistics

‎04-07-Pipeline-ways-to-run-a-pipeline.Rmd‎

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Web-based:
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- Laxy.io / Degust
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- Galaxy Australia
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- <a href="https://laxy.io" target="_blank">laxy.io</a> &rarr; <a href="https://degust.erc.monash.edu/" target="_blank">Degust</a>
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- <a href="https://usegalaxy.org.au/" target="_blank">Galaxy Australia</a>
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Command line:
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- M3 / Monarch HPC clusters (eg using [https://nf-co.re/rnaseq](nf-co.re/rnaseq) )
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- M3 HPC cluster (eg using [https://nf-co.re/rnaseq](nf-co.re/rnaseq) )
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- NeCTAR virtual machine (~32 - 64Gb RAM)
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- Your laptop .. _while not recommended, you may be able to do this for smaller genomes (bacteria, yeast), and larger genomes if using the Salmon pseudo-mapping protocol_

‎files/multiqc/SRP062287_mouse_ref/multiqc_data/junction_saturation_known.txt‎

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Sample 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0
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‎files/multiqc/SRP062287_mouse_ref/multiqc_data/junction_saturation_novel.txt‎

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Sample 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0
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‎files/multiqc/SRP062287_mouse_ref/multiqc_data/mqc_cutadapt_filtered_reads_plot_1.txt‎

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Sample Reads passing filters
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SRR2155413 9113877.0
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SRR2155414 30581575.0
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SRR2155415 29100275.0
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SRR2155416 20318349.0
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SRR2155417 38665207.0
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SRR2155418 29079084.0
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SRR2155419 14980598.0
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SRR2155420 37064178.0
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SRR2155421 22017473.0
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SRR2155422 20836789.0
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SRR2155423 30541349.0
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SRR2155424 26622255.0

‎files/multiqc/SRP062287_mouse_ref/multiqc_data/mqc_cutadapt_trimmed_sequences_plot_3_Counts.txt‎

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Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101
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SRR2155413 - Adapter 1 2073799 721262 194333 45472 16547 9350 7209 6177 5874 4826 4006 3391 2472 2417 2140 1951 2079 1781 957 993 854 695 629 513 557 425 411 431 356 419 182 175 185 199 240 191 169 206 219 169 174 124 133 139 209 239 93 117 133 119 148 259 251 71 83 87 151 194 174 116 168 186 369 290 102 146 249 419 676 638 131 38 17 13 9 6 9 10 9 7 12 8 5 8 6 13 10 11 7 3 4 11 5 12 3 8 7 12 2 5 7
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SRR2155414 - Adapter 1 6859894 2383542 635742 150156 54730 29412 24424 21325 19300 16550 13181 12507 9010 8802 7285 7032 6704 5653 3455 3405 2940 2764 2321 2045 1979 1505 1543 1637 1334 1333 770 819 734 777 702 735 961 1058 445 506 463 428 459 466 656 705 401 416 430 414 466 724 669 283 295 363 494 589 527 454 576 652 1212 1050 523 609 995 1695 2568 2577 687 207 105 56 46 33 25 28 25 26 35 35 15 28 24 24 21 27 29 24 21 49 28 19 14 24 24 43 21 22 75
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SRR2155415 - Adapter 1 6563948 2272436 603445 141259 50332 26366 20582 18229 17230 13427 11154 10072 8036 7211 6648 6054 5916 4982 3146 2998 2634 2271 1945 1732 1826 1289 1494 1761 1416 1505 758 828 792 1074 886 788 775 604 660 779 711 547 670 681 1083 1148 618 518 558 579 663 1284 1181 392 418 558 801 909 965 682 866 1137 2417 1661 654 836 1707 3031 4796 5378 960 345 107 67 39 30 25 25 41 32 58 39 22 43 29 31 31 29 23 30 34 59 26 27 20 31 36 41 28 46 232
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SRR2155416 - Adapter 1 4594362 1608513 426661 100483 36617 19675 15291 13516 12545 10358 8274 7723 5817 5390 4864 4312 4635 3896 2118 2232 1827 1654 1465 1279 1196 955 1016 998 896 888 449 503 447 492 754 616 489 367 404 275 263 316 333 344 605 589 223 240 293 234 334 658 464 140 190 219 330 382 375 255 338 371 718 514 229 255 499 807 1165 1049 246 110 38 20 24 16 11 16 19 26 28 17 16 20 19 22 18 12 18 15 14 24 9 12 9 10 7 22 9 10 19
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SRR2155417 - Adapter 1 8690340 3023193 815323 195337 74229 41805 33412 29702 28329 22883 18559 16531 13216 12185 10424 10486 9926 8791 4845 4735 4286 3619 3226 3018 2897 2264 2086 2047 1869 1871 1068 1062 893 1065 798 830 823 704 760 638 634 672 729 640 1000 901 445 480 469 506 534 978 818 283 332 421 537 637 672 530 609 650 1287 1040 452 526 886 1526 2271 2603 765 273 121 68 43 31 35 23 28 36 38 33 19 44 35 40 30 29 34 30 22 58 33 17 19 23 31 70 25 18 37
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SRR2155418 - Adapter 1 6586868 2273257 596894 137344 45587 22235 17422 14502 13584 11352 9026 8208 6162 5861 4694 4391 4286 3660 2190 2185 1916 1709 1469 1322 1237 1056 1109 1174 954 984 507 609 478 624 575 524 524 414 463 524 390 488 469 498 735 627 369 331 404 325 433 766 620 228 232 288 448 479 525 369 485 462 969 678 327 344 678 1066 1711 1777 468 174 73 39 34 22 34 14 18 37 41 20 15 29 22 31 21 15 29 22 21 43 28 7 14 21 12 36 16 11 33
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SRR2155419 - Adapter 1 3399067 1185108 320090 77703 30611 18343 14936 13201 12158 9927 8202 7257 5736 5365 4690 4376 4427 3947 2077 2023 1796 1604 1471 1328 1225 957 951 923 803 905 425 495 414 520 409 358 368 304 323 264 264 248 274 269 382 348 214 217 232 204 260 417 370 145 160 197 250 316 309 257 282 408 691 570 295 322 586 868 1153 977 185 74 32 16 18 7 14 8 10 5 29 15 13 23 5 13 15 13 14 10 21 15 14 13 4 8 7 20 6 9 30
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SRR2155420 - Adapter 1 8308358 2897491 785706 192783 75939 45009 36259 32092 30754 24734 20168 18498 14364 13416 11780 11121 10667 9040 5764 5338 4636 4080 3575 3245 3045 2352 2285 2425 1930 2051 1038 1103 924 1087 961 1024 844 1027 863 676 652 665 764 727 1207 995 482 473 499 503 516 1084 779 287 320 441 591 671 643 454 596 657 1161 964 392 479 773 1488 2340 2703 826 274 121 81 43 25 21 23 23 26 53 24 25 29 31 42 32 32 20 34 23 58 25 19 15 27 26 53 31 17 54
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SRR2155421 - Adapter 1 4979546 1719313 456809 105514 36960 18867 14057 12639 12227 9490 7921 6832 5334 4937 4251 4176 4316 3647 1820 1895 1731 1383 1296 1247 1148 913 860 883 935 889 506 466 443 640 428 487 410 337 559 382 391 315 360 346 635 445 168 165 190 185 191 559 310 94 117 172 269 245 234 139 199 178 441 236 79 104 177 346 625 720 249 98 40 33 25 12 16 16 9 16 32 22 16 19 14 23 11 17 15 13 16 31 13 12 10 3 16 27 9 7 15
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SRR2155422 - Adapter 1 4676068 1650504 433351 100692 36696 20019 15799 13516 12526 10617 8495 7795 5982 5765 4956 4997 4188 3706 2062 2195 1922 1701 1430 1315 1287 974 1023 1032 907 952 465 489 445 557 498 520 541 480 450 489 527 436 489 535 901 737 299 306 347 316 474 837 600 200 242 308 431 519 456 386 441 658 1038 794 452 440 839 1192 1655 1355 361 101 53 35 35 15 18 19 27 12 35 19 9 25 17 31 16 15 18 12 19 27 8 9 9 15 14 26 11 18 35
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SRR2155423 - Adapter 1 6860628 2385928 651183 158989 63282 37991 31458 27085 24699 21147 16943 15775 11974 11231 9709 9282 8760 7592 4616 4427 3681 3433 2964 2653 2373 1947 1855 1834 1663 1618 891 927 773 862 792 863 781 777 715 655 703 651 662 694 1079 912 462 461 489 458 520 1025 794 305 323 396 567 616 660 473 607 616 1313 1127 402 497 789 1438 2202 2509 795 319 119 62 35 31 20 19 22 26 35 13 26 29 27 39 27 24 22 26 21 45 24 15 23 21 20 32 13 19 37
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SRR2155424 - Adapter 1 5983523 2087608 555260 130455 48078 25852 20912 17882 16688 13955 11054 10425 7497 7079 5885 5288 5478 4543 2724 2658 2316 2028 1751 1622 1494 1228 1102 1250 1017 1040 574 556 482 567 647 537 521 497 404 400 462 407 418 464 683 618 309 325 313 307 360 712 587 212 226 271 384 495 490 325 435 460 884 756 247 343 581 1036 1639 1845 408 161 59 45 38 35 27 29 27 33 27 24 19 20 30 37 12 25 19 25 14 34 23 14 9 12 25 23 17 15 40

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